Agrobacterium-mediated Transformation Method Research Articles

Agrobacterium-Mediated Transient Transformation of Marchantia Liverworts.

Agrobacterium-mediated transient gene expression is a rapid and useful approach for characterizing functions of gene products in planta. However, the practicability of the method in the model liverwort Marchantia polymorpha has not yet been thoroughly described. Here we report a simple and robust method for Agrobacterium-mediated transient transformation of Marchantia thalli and its applicability. When thalli of M. polymorpha were co-cultured with Agrobacterium tumefaciens carrying β-glucuronidase (GUS) genes, GUS staining was observed primarily in assimilatory filaments and rhizoids. GUS activity was detected 2 days after infection and saturated 3 days after infection. We were able to transiently co-express fluorescently tagged proteins with proper localizations. Furthermore, we demonstrate that our method can be used as a novel pathosystem to study liverwort-bacteria interactions. We also provide evidence that air chambers support bacterial colonization.

Efficient knockout of the phytoene desaturase gene in a hybrid poplar (Populus alba × Populus glandulosa) using the CRISPR/Cas9 system with a single gRNA.

The CRISPR/Cas9 system has been used for genome editing in several plant species; however, there are few reports on its use in trees. Here, CRISPR/Cas9 was used to mutate a target gene in Populus alba × Populus glandulosa hybrid poplars. The hybrid poplar is routinely used in molecular biological studies due to the well-established Agrobacterium-mediated transformation method. A single guide RNA (sgRNA) with reported high mutation efficiency in other popular species was designed with a protospacer adjacent motif sequence for the phytoene desaturase 1 (PagPDS1) gene. The pHSE/Cas9-PagPDS1 sgRNA vector was delivered into hybrid poplar cells using Agrobacterium-mediated transformation. The transgenic plants were propagated and classified them into three groups according to their phenotypes. Among a total of 110 lines of transgenic hybrid poplars, 82 lines showed either an albino or a pale green phenotype, indicating around 74.5% phenotypic mutation efficiency of the PagPDS1 gene. The albino phenotypes were observed when the CRISPR/Cas9-mediated mutations in both PagPDS1 alleles in the transgenic plants. There was no off-target modification of the PagPDS2 gene, which has a potential sgRNA target sequence with two mismatches. The results confirmed that the sgRNA can specifically edit PagPDS1 rather than PagPDS2, indicating that CRISPR/Cas9-mediated genome editing can effectively induce target mutations in the hybrid poplar. This technique will be useful to improve tree quality in hybrid poplars (P. alba × P. glandulosa); for example, by enhancing biomass or stress tolerance.

Agrobacterium-mediated In-planta transformation of bread wheat (Triticum aestivum L.)

Agrobacterium-mediated in-planta transformation method allows efficient plant transformation without tissue culture. In the present study, a tissue culture independent protocol for floral transformation in bread wheat was developed. Three bread wheat genotypes namely, HD 2967, HD 3086 and Bobwhite were used for floral transformation. At pre-anthesis stage, clipped florets were sprayed with inoculation medium containing Agrobacterium tumefaciens strain EHA 105 culture harboring binary vector pCAMBIA1301-p35S:hptII-p35S:gus and allowed to grow till maturity. Positive transformants were confirmed through antibiotic selection, PCR analysis and southern blotting. The method led to generation of transformation events in Bobwhite and HD 2967 wheat genotypes. The transformation efficiency of T1 transgenic plants was higher in Bobwhite as compared to HD 2967. Floral spray method of transformation developed in the present study is efficient for development of transgenic wheat and can be used for improvement of wheat using transgenic approach.

Establishment of A Simple and Efficient Agrobacterium-mediated Genetic Transformation System to Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

Chinese cabbage, belonging to Brassica rapa species, is an important vegetable in Eastern Asia. It is well known that Chinese cabbage is quite recalcitrant to genetic transformation and the transgenic frequency is generally low. The lack of an efficient and stable genetic transformation system for Chinese cabbage has largely limited related gene functional studies. In this study, we firstly developed a regeneration system for Chinese cabbage by optimizing numerous factors, with 93.50% regeneration rate on average. Based on this, a simple and efficient Agrobacterium-mediated genetic transformation method was established, without pre-culture procedure and concentration adjustment of hormone and AgNO3 in co-cultivation and selection media. Using this system, transformants could be obtained within 3.5–4.0 months. Average transformation frequency is up to 10.83%. The establishment of this simple and efficient genetic transformation method paved the way for further gene editing and functional studies in Chinese cabbage.

Effects of vacuum infiltration, Agrobacterium cell density and acetosyringone concentration on Agrobacterium-mediated transformation of bread wheat

The recent advances in genome-wide transcriptome analysis have enabled researchers to identify new genes and manipulate the genome through novel genetic engineering methods to improve plant tolerance to different stresses. We conducted a genome-wide transcriptome analysis to determine tomato genes affecting the salt stress response. The results showed that among the 5784 genes that were responsive to salt stress, 103 genes (1.8%) encode transcription factors, of which 69 (1.2%) were upregulated and 34 (0.6%) were downregulated. The largest group of genes upregulated in response to salt stress (17 genes) is related to the ethylene response transcription factors (ERF) family. Specifically, it was found that the gene JERF1, encoding ERF in tomato, is upregulated in response to salt stress. In the present study we developed a new Agrobacterium-mediated transformation method to deliver the JERF1 gene to mature wheat embryos. It was investigated whether the factors vacuum infiltration, Agrobacterium cell density (OD600 1.0 and its dilutions 1:20, 2:20 and 3:20), the acetosyringone concentration (0, 200 and 400 μM) and the interaction between these factors affect transformation. To this end, Agrobacterium carrying pGWB14-JERF1 was injected into soaked wheat seeds, and T0 transgenic plants were obtained in the greenhouse. Molecular analysis of T0 plants was performed. The highest transformation efficiency was obtained under vacuum infiltration, 200 μM acetosyringone and 1:20 dilution. Notably, we used the in planta transformation method to overcome some of the problems of traditional wheat transformation methods e.g., sterile conditions, recalcitrant regeneration, time-constraints and somaclonal variations.

Development of an efficient in-planta Agrobacterium-mediated transformation method for Iranian purslane (Portulaca oleracea L.) using sonication and vacuum infiltration

Purslane (Portulaca oleracea L.), a valuable medicinal herb, is used as a source of pharmaceutical components such as flavonoids, alkaloids, fatty acids, terpenoids, and sterols. Regeneration of transgenic purslane plantlets from transformed cells is a time-consuming procedure and needs hard work. In this study, in-planta transformation of purslane, using sonication and vacuum infiltration, is reported. The purslane seeds were infected through Agrobacterium tumefaciens strain LBA4404 having the uidA gene on pBI121 vector. Effective selection of transformants was performed by supplementing MS media with 250 mg l−1 kanamycin. Several factors affecting the in-planta procedure including pre-culture duration, acetosyringone dose, sonication, and vacuum infiltration duration were investigated. The results demonstrated that the highest number of GUS-positive purslane plantlets was obtained when the pre-cultured seeds were sonicated and vacuum-infiltered for 2 min in agro-bacterial cell suspension, and then co-cultivated in MS media having 100 µM acetosyringone. The integration and expression of uidA gene in transgenic purslane was successfully corroborated by southern blot and GUS histochemical analyses.

Development of a Transformable Fast-Flowering Mini-Maize as a Tool for Maize Gene Editing.

Maize (Zea mays ssp. mays) is a popular genetic model due to its ease of crossing, well-established toolkits, and its status as a major global food crop. Recent technology developments for precise manipulation of the genome are further impacting both basic biological research and biotechnological application in agriculture. Crop gene editing often requires a process of genetic transformation in which the editing reagents are introduced into plant cells. In maize, this procedure is well-established for a limited number of public lines that are amenable for genetic transformation. Fast-Flowering Mini-Maize (FFMM) lines A and B were recently developed as an open-source tool for maize research by reducing the space requirements and the generation time. Neither line of FFMM were competent for genetic transformation using traditional protocols, a necessity to its status as a complete toolkit for public maize genetic research. Here we report the development of new lines of FFMM that have been bred for amenability to genetic transformation. By hybridizing a transformable maize genotype high Type-II callus parent A (Hi-II A) with line A of FFMM, we introgressed the ability to form embryogenic callus from Hi-II A into the FFMM-A genetic background. Through multiple generations of iterative self-hybridization or doubled-haploid method, we established maize lines that have a strong ability to produce embryogenic callus from immature embryos and maintain resemblance to FFMM-A in flowering time and stature. Using an Agrobacterium-mediated standard transformation method, we successfully introduced the CRISPR-Cas9 reagents into immature embryos and generated transgenic and mutant lines displaying the expected mutant phenotypes and genotypes. The transformation frequencies of the tested genotypes, defined as the numbers of transgenic event producing T1 seeds per 100 infected embryos, ranged from 0 to 17.1%. Approximately 80% of transgenic plants analyzed in this study showed various mutation patterns at the target site. The transformable FFMM line, FFMM-AT, can serve as a useful genetic and genomic resource for the maize community.

Ectopic expression of poplar gene PsnERF138 in tobacco confers salt stress tolerance and growth advantages.

The AP2/ERF family is one of the largest plant-specific transcription factors and plays a vital role in plant growth and stress response. In this study, PsnERF138 was cloned from Populus alba×Populus glandulosa and transformed into tobacco using the Agrobacterium-mediated transformation method. PsnERF138 was localized in the nucleus through subcellular localization assay in tobacco. Under normal conditions, the root lengths of PsnERF138 transgenic lines were much longer than those of wild type. Under salt stress, the transgenic tobacco lines over-expressing PsnERF138 showed a significant increase in seed germination rate, plant height, and root length, compared to control plants. In addition, the transgenic tobacco lines displayed some advantages at the physiological level, such as higher superoxide dismutase (SOD) activity, peroxidase (POD) activity, proline content, and lower malondialdehyde (MDA) content, as compared to those in the control plants. Histochemical staining also showed that the transgenic tobacco lines had lower reactive oxygen species (ROS) accumulation, compared to control plants under salt stress. The combined results indicate that poplar PsnERF138 plays a contributing role in augmenting salt tolerance and conferring multiple growth advantages as being overexpressed in tobacco.

Agrobacterium-Mediated Transformation Improvement in Tomato

In this article, we describe a highly efficient and reproducible method of Agrobacterium-mediated transformation protocol for several gene transformations (cry1Ab, rolB, and PhMV) in tomatoes. Tomato fruit is rich in minerals, essential amino acids, sugar, vitamin B, iron, phosphorus, and dietary fiber. Tomato Crop production has some major problem which includes early ripening, reduction in fruit shelf life and devastating diseases that caused by viruses, bacteria, fungi, and nematodes, etc. The study aims to use the Agrobacterium-mediated transformation method to improve the tomato shelf life and make tomato plants resistant to such types of diseases caused by bacteria, viruses, and fungi, etc. Tomato was transformed via Agrobacterium tumefaciens strain LBA4404 harbouring Cry1Ab synthetic gene for toxin proteins. And same strain was inserted with another CP of PhMV that cloned into the binary vector pBI 121 for resistance to PhMV infection, and strain GV3101 harbouring the binary vector pICBV19 for reducing the expression of vis1 gene. Tomato seed culture on MS media after sterilization with 75% ethanol and distilled water. Plantlet root formation was on different kanamycin MS mediums. Constricted plasmid vectors with a gene if interest allowed to be transferred into Agrobacterium tumefaciens using the freeze-thaw method. The engineered strain suspension was centrifuged and re-suspended with MS media. Tomato seeds were obtained, then 0.5 cm per cotyledon, and put into the Agrobacterium suspension and co-cultivated and incubated and a transgenic plant, analysis was carried out using PCR, real-time Reanalysis, Northern blot analysis, and statistical analysis. For tomato varieties, the protocol developed showed very high transformation efficiency. The optimized transformation process is simple, effective and no need for tobacco, Petunia, tomato suspension feeder layer, or acetosyringone as chemical transduction.

Stable and Efficient Agrobacterium-Mediated Genetic Transformation of Larch Using Embryogenic Callus.

Larix olgensis or larch is an economically important coniferous tree species with rapid growth in the early stages, strong adaptability, and a short time to harvest. The genetic improvement of larch has garnered considerable attention in recent years for reclaiming timber forests. However, traditional breeding methods are largely ineffective for achieving rapid genetic improvement of L. olgensis. Studies show that the efficiency of plant regeneration can be improved by optimizing somatic embryogenesis. On this basis, we devised a stable, fast and efficient Agrobacterium-mediated genetic transformation method using suspended embryogenic calluses as explants and β-glucuronidase as the reporter. We evaluated the effects of the Agrobacterium load, co-culture period, and addition of acetosyringone and transformant screening antibiotic on the transformation efficiency. In addition, we tested the pCAMBIA 1300-PtHCA 2-1 promoter-GUS binary expression vector, which contains the GUS gene ORF under the control of Populus trichocarpa high cambial activity PtHCA 2-1 promoter, and observed the tissue-specific expression of the GUS gene in the somatic embryos of transgenic larch. This novel technique can not only accelerate the generation of superior transgenic strains of L. olgensis but also aid in future gene functional studies.

Overexpression of Chitinase gene enhances resistance to Colletotrichum gloeosporioides and Alternaria alternata in apple (Malus×domestica)

Apple is a cultivated fruit tree, worldwide. However, the growth and development of apple have been increasingly affected by fungal disease. Chitinase is one of the most important defensive proteins in plant. Their biological function and disease resistance mechanism, including apple, is rarely reported. In our previous study, we found that autotetraploid 'Hanfu' apple is more resistant to fungal disease than diploid apple. In addition, RNA sequencing (RNA-seq) analysis revealed that the expression level of the MdCHI1 gene was significantly up-regulated in the autotetraploid apple. In this work, we isolated CHI1 from 'Hanfu' apple, and the quantitative real-time PCR (qRT-PCR) analyses demonstrated that MdCHI1 was highly expressed in leaves of apple and was rapidly induced by MeJA. Then, we obtained 4 lines of MdCHI1-OE by Agrobacterium-mediated transformation method. The MdCHI1-OE plants were more resistant to fungal diseases caused by Colletotrichum gloeosporioides and Alternaria alternata, moreover the chitinase activity in MdCHI1-OE plants was greater than that of 'GL-3′ (WT) plants. Taken together, the data provide insights that MdCHI1 as an important role in the fungal disease resistance of apple.

Genotype-Independent Transformation and Genome Editing of Brassica napus Using a Novel Explant Material.

Agrobacterium-mediated transformation of canola (Brassica napus) via hypocotyl segments has been a commonly used method for the past 30 years. While the hypocotyl-based method is well-established, it is not readily adapted to elite germplasm and the prolonged process is not ideal for a production transformation setting. We developed an Agrobacterium-mediated transformation method based on epicotyl and higher stem (internodal) segments that is efficient, rapid and amenable for high-throughput transformation and genome editing. The method has been successfully implemented in multiple canola genotypes. The method appears to be genotype-independent, with varying transformation efficiencies. Internodal segment transformation was used to generate transgenic events as well as CRISPR-Cas9-mediated frameshift gene knockouts.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2-3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

An efficient Agrobacterium-mediated transformation method using hypocotyl as explants for Brassica napus

Rapeseed (Brassica napus) is an important oil crop that supplies a considerable amount of global vegetable oil production. Genetic transformation system is important to gene functional analysis and molecular breeding. Here, an efficient Agrobacterium-mediated transformation protocol using hypocotyl of rapeseed as explants is described. To develop this protocol, we compared several essential factors that would affect the transformation efficiency, such as Agrobacterium strains, selection marker genes, and genotypes of rapeseed. Comparison of different Agrobacterium strains showed that the GV3101 had higher transformation efficiency than that of C58C1 and EHA105. HPTII, NPTII, and RePAT were used as selection marker genes in tissue culture. The results showed that the transformation efficiency was 3.7–4.8%, 2.2–22.5%, and 1.6–5.9% when the hypocotyl of Westar was infected by GV3101 and screened under hygromycin, kanamycin, and basta, respectively. The transformation efficiency of Westar was the highest and ZS11 was the lowest when five different genotypes of rapeseed (Westar, ZS9, ZS11, GY284, and WH3417) were infected by GV3101. Using this protocol, it will take 8–10 weeks to obtain transgenic plants. This protocol has been used to study gene function in several genotypes of rapeseed in our laboratory. These results indicate that it is efficient to obtain transgenic plant of rapeseed using this protocol.

High transformation efficiency in Arabidopsis using extremely low Agrobacterium inoculum

Agrobacterium-mediated transformation methods have allowed the stable introduction of target genes into the nuclear genomes of recipient plants. Among them, the floral dip approach represents the simplest due to its straightforwardness and high transformation efficiency. In a standard floral dip protocol that most researchers follow, Agrobacterium cells are grown to stationary phase (OD 600≈2.0) in large cultures and resuspended in inoculation medium to OD 600≥0.8. Here, we tested the effects of low Agrobacterium inoculum on transformation rate. Our data revealed that the floral dip method still guarantees a relatively high transformation rate in the Arabidopsis thaliana Col-0 ecotype even with very low Agrobacterium inoculum (OD 600=0.002). Our finding thus simplifies the floral dipping protocol further, which allows transformation with small bacterial culture and enables high-throughput transformation of large numbers of constructs in parallel.

Assessment of biolistic and Agrobacterium-mediated genetic transformation methods in Cenchrus ciliaris

Cenchrus ciliaris, commonly known as buffelgrass, is an apomictic perennial range grass usually grown in arid/semi-arid regions. Because of the difficulties faced in conventional breeding of this polymorphic polyploid grass, the development of an efficient protocol for genetic transformation is warranted. Such a protocol would enable functional genomic studies needed to elucidate the mechanism of apomixis in this species. The embryogenic calli for genetic transformation experiments were obtained by in vitro culture of the immature inflorescences of buffelgrass cv. IGFRI-3108. Four developmental stages of immature inflorescences were compared for callus induction, and the most mature stage produced most callus. Embryogenic calli were bombarded at three distances, 6 cm (L1), 9 cm (L2), or 12 cm (L3) with a marker gene uid A present in pCAMBIA1301, under the same vacuum (85 kPa) and at constant pressure (900 psi). The Agrobacterium-mediated genetic transformation was also performed using the same construct. Transient and stable expression as well as PCR amplification of the GUS gene was used for comparative analysis as well as for validation of the transformants. Transient GUS expression was present in a significantly higher percentage of bombarded calli (56.33%) than of Agrobacterium treated calli (11.17%), but the number of GUS positive cells per callus was similar. Among the three different bombardment distances, transient GUS expression was highest at L2, but stable GUS expression was highest at L1. Shoot development from the bombarded calli could be accomplished, which failed from the Agrobacterium-mediated transformed calli. Thus, the results indicate that C. ciliaris cv. IGFRI-3108 can be successfully transformed through Biolistic particle bombardment, while Agrobacterium-mediated transformation requires further optimization of transformation protocols.

Optimization of multiplexed CRISPR/Cas9 system for highly efficient genome editing in Setaria viridis.

In recent years, Setaria viridis has been developed as a model plant to better understand the C4 photosynthetic pathway in major crops. With the increasing availability of genomic resources for S. viridis research, highly efficient genome editing technologies are needed to create genetic variation resources for functional genomics. Here, we developed a protoplast assay to rapidly optimize the multiplexed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system in S. viridis. Targeted mutagenesis efficiency was further improved by an average of 1.4-fold with the exonuclease, Trex2. Distinctive mutation profiles were found in the Cas9_Trex2 samples, with 94% of deletions larger than 10bp, and essentially no insertions at all tested target sites. Further analyses indicated that 52.2% of deletions induced by Cas9_Trex2, as opposed to 3.5% by Cas9 alone, were repaired through microhomology-mediated end joining (MMEJ) rather than the canonical non-homologous end joining DNA repair pathway. Combined with a robust Agrobacterium-mediated transformation method with more than 90% efficiency, the multiplex CRISPR/Cas9_Trex2 system was demonstrated to induce targeted mutations in two tightly linked genes, svDrm1a and svDrm1b, at a frequency ranging from 73% to 100% in T0 plants. These mutations were transmitted to at least 60% of the transgene-free T1 plants, with 33% of them containing bi-allelic or homozygous mutations in both genes. This highly efficient multiplex CRISPR/Cas9_Trex2 system makes it possible to create a large mutant resource for S. viridis in a rapid and high throughput manner, and has the potential to be widely applicable in achieving more predictable and deletion-only MMEJ-mediated mutations in many plant species.

Soybean Embryonic Axis Transformation: Combining Biolistic and Agrobacterium-Mediated Protocols to Overcome Typical Complications of In Vitro Plant Regeneration.

The first successful attempt to generate genetically modified plants expressing a transgene was preformed via T-DNA-based gene transfer employing Agrobacterium tumefaciens-mediated genetic transformation. Limitations over infectivity and in vitro tissue culture led to the development of other DNA delivery systems, such as the biolistic method. Herein, we developed a new one-step protocol for transgenic soybean recovery by combining the two different transformation methods. This protocol comprises the following steps: agrobacterial preparation, seed sterilization, soybean embryo excision, shoot-cell injury by tungsten-microparticle bombardment, A. tumefaciens-mediated transformation, embryo co-cultivation in vitro, and selection of transgenic plants. This protocol can be completed in approximately 30–40 weeks. The average efficiency of producing transgenic soybean germlines using this protocol was 9.84%, similar to other previously described protocols. However, we introduced a more cost-effective, more straightforward and shorter methodology for transgenic plant recovery, which allows co-cultivation and plant regeneration in a single step, decreasing the chances of contamination and making the manipulation easier. Finally, as a hallmark, our protocol does not generate plant chimeras, in contrast to traditional plant regeneration protocols applied in other Agrobacterium-mediated transformation methods. Therefore, this new approach of plant transformation is applicable for studies of gene function and the production of transgenic cultivars carrying different traits for precision-breeding programs.

An efficient protocol for Agrobacterium-mediated genetic transformation of Antirrhinum majus

Snapdragon (Antirrhinum majus L.) is a popular ornamental and model plant species, and the recently released reference genome could greatly boost its utilization in fundamental research. However, the lack of an efficient genetic transformation system is still a major limiting factor for its full application in genetic and molecular studies. In this study, a simple method for quick regeneration and efficient Agrobacterium-mediated transformation of snapdragon was developed. Cotyledon petiole and hypocotyl explants derived from two-week-old seedlings were cultured on MS media supplemented with 2 mg/L zeatin (ZT), 0.2 mg/L 1-naphthaleneacetic acid (NAA), and 2 mg/L AgNO3, and adventitious shoots were regenerated through organogenesis with an average regeneration of 48.00% and 41.33%, respectively. By contrast, the regeneration frequency was only 22.67% for cotyledon petiole and 25.67% for hypocotyl explants in the absence of AgNO3. Moreover, the application of AgNO3 promoted indirect shoot organogenesis, while direct shoot organogenesis occurred in the absence of AgNO3 from both hypocotyl or cotyledon petiole explants. Agrobacterium-mediated genetic transformation systems were developed with this high-efficient regeneration system. The transformation efficiency has been improved from 0 to 1% through the direct shoot organogenesis to 3 to 4% via the indirect shoot organogenesis. This efficient regeneration and genetic transformation method could be important for future use of snapdragon as a model plant to address some fundamental questions which are hard to be solved by using other model plant species, and to accelerate the breeding process through CRISPR/Cas9 genome editing. Genetic transformation of snapdragon was significantly improved by applying AgNO3 to induce indirect organogenesis, which will substantially facilitate its fundamental research as well as molecular breeding.

Overexpression of a Eutrema salsugineum phosphate transporter gene EsPHT1;4 enhances tolerance to low phosphorus stress in soybean.

To enhance Pi absorption and utilization efficiency of soybean, a member of PHT1 gene family was isolated and characterized from E. salsugineum, which was a homologous gene of AtPHT1;4 and consequently designated as EsPHT1;4. Quantitative real-time PCR (qRT-PCR) analysis showed that the transcript level of EsPHT1;4 significantly increased both in roots and leaves of E. salsugineum under Pi deficient conditions. Furthermore, EsPHT1;4 was transferred to soybean cultivar "YD22" using an Agrobacterium-mediated cotyledonary-node transformation method. Overexpression of EsPHT1;4 in soybean not only promoted the increase of plant biomass and yield of transgenic plants upon low P stress, but also increased the accumulation and transportation of Pi from roots to leaves in the transgenic soybean lines. EsPHT1;4 was critical for controlling the accumulation and translocation of Pi in plants, and can be subsequently used as an effective foreign gene for the improvement of P use efficiency of crops by genetic manipulation.